Mitochondrial enhancement of cells

ABSTRACT

Certain embodiments disclosed herein include, but are not limited to, at least one of compositions, methods, devices, systems, kits, or products regarding rejuvenation or preservation of stem cells. Certain embodiments disclosed herein include, but are not limited to, methods of modifying stem cells, or methods of administering modified stem cells to at least one biological tissue.

RELATED APPLICATIONS

For purposes of the USPTO extra-statutory requirements, the presentapplication constitutes a continuation-in-part of U.S. patentapplication Ser. No. 12/925,849, entitled MITOCHONDRIAL ENHANCEMENT OFCELLS, naming Roderick A. Hyde and Lowell L. Wood, Jr. as inventors,filed 28 Oct. 2010, which is currently co-pending, or is an applicationof which a currently co-pending application is entitled to the benefitof the filing date.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is related to and claims the benefit of theearliest available effective filing date(s) from the following listedapplication(s) (the “Related Applications”) (e.g., claims earliestavailable priority dates for other than provisional patent applicationsor claims benefits under 35 USC §119(e) for provisional patentapplications, for any and all parent, grandparent, great-grandparent,etc. applications of the Related Application(s)). All subject matter ofthe Related Applications and of any and all parent, grandparent,great-grandparent, etc. applications of the Related Applications isincorporated herein by reference to the extent such subject matter isnot inconsistent herewith.

The United States Patent Office (USPTO) has published a notice to theeffect that the USPTO's computer programs require that patent applicantsreference both a serial number and indicate whether an application is acontinuation or continuation-in-part. Stephen G. Kunin, Benefit ofPrior-Filed Application, USPTO Official Gazette Mar. 18, 2003, availableat http://www.uspto.gov/web/offices/com/sol/og/2003/week11/patbene.htm.The present Applicant Entity (hereinafter “Applicant”) has providedabove a specific reference to the application(s) from which priority isbeing claimed as recited by statute. Applicant understands that thestatute is unambiguous in its specific reference language and does notrequire either a serial number or any characterization, such as“continuation” or “continuation-in-part,” for claiming priority to U.S.patent applications. Notwithstanding the foregoing, Applicantunderstands that the USPTO's computer programs have certain data entryrequirements, and hence Applicant is designating the present applicationas a continuation-in-part of its parent applications as set forth above,but expressly points out that such designations are not to be construedin any way as any type of commentary and/or admission as to whether ornot the present application contains any new matter in addition to thematter of its parent application(s).

All subject matter of the Related Applications and of any and allparent, grandparent, great-grandparent, etc. applications of the RelatedApplications is incorporated herein by reference to the extent suchsubject matter is not inconsistent herewith.

SUMMARY

Disclosed herein include embodiments relating to compositions, methods,delivery devices, computer systems, program products, andcomputer-implemented methods related to modified stem cells.

The foregoing summary is illustrative only and is not intended to be inany way limiting. In addition to the illustrative aspects, embodiments,and features described above, further aspects, embodiments, and featureswill become apparent by reference to the drawings and the followingdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a partial view of an example of a biological cell,including various components.

FIG. 2 illustrates a partial view of an example of a mitochondrion,including various components.

FIG. 3 illustrates a partial view of a particular embodiment of amethod.

FIG. 4 illustrates a partial view of a particular embodiment of themethod of FIG. 3.

FIG. 5 illustrates a partial view of a particular embodiment of themethod of FIG. 3.

FIG. 6 illustrates a partial view of a particular embodiment of themethod of FIG. 3.

FIG. 7 illustrates a partial view of a particular embodiment of themethod of FIG. 3.

FIG. 8 illustrates a partial view of a particular embodiment of themethod of FIG. 3.

FIG. 9 illustrates a partial view of a particular embodiment of themethod of FIG. 3.

FIG. 10 illustrates a partial view of a particular embodiment of themethod of FIG. 3.

FIG. 11 illustrates a partial view of a particular embodiment of themethod of FIG. 3.

FIG. 12 illustrates a partial view of a particular embodiment of acomputer program product.

FIG. 13 illustrates a partial view of a particular embodiment of asystem.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented here.

Certain aspects described herein relate to modifying a eukaryotic cellby providing at least one exogenous cellular component. In anembodiment, the exogenous cellular component includes at least one of anexogenous mitochondria, exogenous mitochondrial DNA, DNA of the cellnucleus, or exogenous cell nucleus to the eukaryotic cell. In anembodiment, at least one of the exogenous mitochondria, exogenousmitochondrial DNA, or exogenous cell nucleus is derived from amaternally genetically related cell.

Mitochondria are controlled by a dual genome system (particularly inhumans) with cooperation between endogenous mitochondrial genes andmitochondrial genes translocated to the nucleus over the course ofevolution. See, for example, Cummins, Human Rep. Update vol. 7, no. 2,pp. 217-228 (2001), which is incorporated herein by reference.Mitochondria likely play a role in aging, apoptosis, metabolism, andmany diseases. The mitochondrial genome is highly compact, with littletolerance for mutations. Id. Thus, the ability to construct cells, cellsystems, or animal embryos with maternally genetically relatedmitochondria, mitochondrial DNA, or cell nucleus, will provide powerfultools for biological tissue generation, regeneration, and alleviation ofa subject from diseases.

The function of mitochondria by both genes of the nucleus andmitochondrial genes can result in conflict, under certain conditions.Id. For example, genome imprinting occurs where certain genesinfluencing placental and embryonic development are expressed orsuppressed according to whether they pass through paternal or maternalgametogenesis. Id.

The complete mitochondrial sequence is known for more than 58 chordateand 29 non-chordate species. Id. The genome is generally a closedcircular molecule with little redundancy, although linear forms withtelomere-like terminations are known. Id. Mitochondrial DNA is generallytightly linked to the electron transport system and is vulnerable todamage since certain components mutate up to 100 times more rapidly thanDNA of the cell nucleus. Id.

It has been published that cell lines can be constructed devoid ofendogenous mitochondrial DNA (mtDNA), and re-populated with xenogenicmitochondrial lineages, which demonstrates that survival ofmitochondrial genotypes is dependent on the nucleus background. Id.Furthermore, it has been published that rat mtDNA can restoretranslation but not respiration in mtDNA-depleted mouse cell lines. Id.Thus, the cell functions more efficiently when there is compatibilitybetween the DNA of the nucleus and mitochondrial genes of the cell.

Ordinarily, cells contain only one type of mitochondrial DNA. It hasbeen published that following cytoplasmic transfer or transfer of a cellnucleus in animal cells, multiple different types of mitochondrial DNAor mismatching of mitochondrial DNA and DNA of the nucleus can result.See, for example, Spikings, et al. Hum. Rep. Update vol. 12, no. 4, pp.401-415 (2006), which is incorporated herein by reference.

For example, mitochondrial cristae are the site of the electron transferchain, the last stage in cellular respiration where oxidativephosphorylation takes place. Id. This aerobic process allows furthermetabolism of the products of anaerobic glycolysis and the citric acidcycle to produce carbon dioxide and water, with the subsequent releaseof 32 molecules of ATP. Id. The components of the electron transferchain are encoded by both chromosomal and mitochondrial DNA (mtDNA),with all components being required for efficient function.

In an embodiment, the eukaryotic cell includes a cybrid formed by thegenome of the nucleus from one source (e.g., endogenous) and themitochondrial genome from another source (e.g., from a maternallygenetically related cell). For example, Rho-zero cells (cells devoid ofmitochondrial DNA) can be repopulated with exogenous mitochondrial DNA,resulting in transmitochondrial cybrids. Id. Alternatively, cells can befused or chemically combined, resulting in cybrid or hybrid cells.

It has been published that genome of the nucleus and mitochondrialgenomes interact and communicate in eukaryotic cells. See, for example,Poyton and McEwen, Ann. Rev. Biochem.; Abstract; vol. 65, pp. 563-607(1996), which is incorporated herein by reference. For example, thegenome of the nucleus encodes essential subunit polypeptides utilized inmitochondrial proteins, and is important for several reasons, includingthe regulation of oxidative energy production. Secondly, theycollaborate in the synthesis and assembly of the proteins, whichrequires the bidirectional flow of information between the nucleus andthe mitochondria. Id. In other published examples, the genome of thenucleus encodes proteins that account for about 90% of the protein massof the mitochondria, including in all four mitochondrial compartments(inner and outer mitochondrial membranes, matrix, and intermembranespace). Id. By contrast, the mitochondrial genome specifies only a fewproteins, which reside mainly in the inner mitochondrial membrane. Id.The mitochondrial genome also encodes RNA molecules that co-assemblewith proteins encoded in the nucleus. Id.

It has been published that mitochondrial gene products are components ofmultimeric protein complexes that contain nucleus-encoded components aswell. Id. For example, components common to all eukaryotic cells thatutilize components encoded by both DNA of the nucleus, as well asmitochondrial DNA include coenzyme Q cytochrome c reductase, cytochromec oxidase, F₁F₀ ATPase, and the mitochondrial ribosome. Id. In someeukaryotic cells, NADH dehydrogenase, small and large ribosomalsubunits, RNase-P, and RNA splicing enzymes include chimeric components,as well. Id.

With regard to regulation of biosynthesis of mitochondrial proteins,trans-acting genes of the nucleus serve to modulate either the level ofexpression of mitochondrial genes or in the assembly of respiratoryproteins. Id. In certain instances, trans-acting genes of the nucleusserve to modulate either the level of expression of the mitochondrialgenome as a whole, or the expression of individual genes on themitochondrial genome. Id. Furthermore, communication from the genome ofthe nucleus to the mitochondria involves proteins that are translated inthe cytosol and imported into the mitochondria. Id. Communication fromthe mitochondria to the nucleus likely involves metabolic signals andone or more signal transduction pathways that function across the innermitochondrial membrane. Id.

It has also been published that mitochondrial gene expression involvesinteractions between nucleus-coded gene products and the mitochondrialgenome or its gene products at several junctions. For example,transcription of mitochondrial gene expression requires interaction withnucleus-coded gene products, RNA processing, and translation utilizevarious nucleus-coded gene products. Id.

In another example, the proteins encoded by nucleus genes are translatedon cytosolic ribosomes (or ribosomes bound to the outer mitochondrialmembrane), and are imported by the mitochondria posttranslationally,whereas proteins encoded by mitochondrial genes are translated onendogenous mitochondrial ribosomes that are bound to the matrix side ofthe inner membrane, and are inserted into it cotranslationally. Id.

It has been published that the genome of the nucleus exerts influenceover mitochondrial gene expression as well as the import, export, andassembly pathways required for biogenesis of functional mitochondria.Id.

Thus, supplementation or complementation by mitochondrial replacement,mitochondrial DNA replacement, cell nucleus replacement, ornucleocytoplasmic replacement provides a basis for therapy for cellswhose respiration ability is compromised (e.g., due to aging ordisease). In certain instances, at least one of mitochondria,mitochondrial DNA, cell nucleus, or nucleocytoplasm is provided to aeukaryotic cell from a maternally genetically related cell. In somecases, the mitochondria, mitochondrial DNA, cell nucleus, ornucleocytoplasm is provided by way of a carrier (e.g., platelet, lipid,polymeric vehicle, etc.).

In an embodiment, a modified eukaryotic cell comprises at least oneexogenous cellular component from a genetically maternally relatedsource. In an embodiment, the at least one exogenous cellular componentincludes at least one of exogenous mitochondria, exogenous mitochondrialDNA, DNA of the cell nucleus, or exogenous cell nucleus. In anembodiment, the at least one exogenous cellular component is located inat least one intracellular compartment of the modified eukaryotic cell.

In an embodiment, a modified eukaryotic cell includes at least oneexogenous cellular component from a genetically maternally relatedsource.

In an embodiment, the intracellular compartment of a eukaryotic cellincludes at least one of a nucleolus, nucleus, ribosome, vesicle, roughendoplasmic reticulum, Golgi apparatus, cytoskeleton, smooth endoplasmicreticulum, mitochondria, vacuole, cytoplasm, lysosome, or centriole. Inan embodiment, the eukaryotic cell is located in at least one of insitu, in vitro, in vivo, in utero, in planta, in silico, or ex vivo.

In an embodiment, the eukaryotic cell is implantable or transplantable.In an embodiment, the eukaryotic cell is implanted or transplanted intoat least one subject. In an embodiment, the eukaryotic cell is implantedor transplanted into at least one subject subsequent to modification. Inan embodiment, the eukaryotic cell is implanted subsequent tomodification, into the original subject from which it was extracted.

In an embodiment, at least one eukaryotic cell is selected formodification based on one or more eukaryotic cell parameters. In anembodiment, the one or more eukaryotic cell parameters relate to atleast one property of the eukaryotic cell. In an embodiment, the one ormore eukaryotic cell parameters include at least one of eukaryotic cellsize, eukaryotic cell stage, eukaryotic cell quality, health of thesubject from which the eukaryotic cell originates, species of thesubject from which the eukaryotic cell originates, cleavage rate of theeukaryotic cell, metabolic profile of the eukaryotic cell, genomicprofile of the eukaryotic cell, transcriptomic profile of the eukaryoticcell, proteomic profile of the eukaryotic cell, or storage conditions ofthe eukaryotic cell (if previously stored ex vivo). In an embodiment,the storage conditions of the eukaryotic cell include at least one ofduration of storage time, storage temperature, storage size, eukaryoticcell dilution, or storage solution(s).

In an embodiment, a modified eukaryotic cell is produced by the processof providing at least one exogenous cellular component derived from amaternally genetically related eukaryotic cell. In an embodiment, the atleast one exogenous cellular component includes at least one ofexogenous mitochondria, exogenous mitochondrial DNA, or exogenous cellnucleus. In an embodiment, the eukaryotic cell is located in at leastone of in situ, in vitro, in vivo, in utero, in planta, in silico, or exvivo. In an embodiment, the eukaryotic cell is implantable ortransplantable.

In an embodiment, the eukaryotic cell is implanted or transplanted intoat least one subject. In an embodiment, the eukaryotic cell is implantedor transplanted into at least one subject subsequent to modification. Inan embodiment, the at least one subject includes at least one of aplant, alga, or animal. In an embodiment, the at least one subjectincludes at least one of a vertebrate or invertebrate. In an embodiment,the at least one subject includes at least one of an amphibian, mammal,reptile, fish, or bird.

In an embodiment, the at least one subject includes at least one human.In an embodiment, the at least one subject includes at least one plant.In an embodiment, the at least one subject includes at least one of afood crop, ornamental, aquatic plant. In an embodiment, the modifiedeukaryotic cell further comprises one or more of a suspension, mixture,solution, sol, clathrate, colloid, emulsion, microemulsion, aerosol,ointment, capsule, micro-encapsule, powder, tablet, suppository, cream,device, paste, resin, liniment, lotion, ampule, elixir, spray, syrup,foam, pessary, tincture, detection material, polymer, biopolymer,buffer, adjuvant, diluent, lubricant, disintegration agent, suspendingagent, solvent, light-emitting agent, colorimetric agent, glidant,anti-adherent, anti-static agent, surfactant, plasticizer, emulsifyingagent, flavor, gum, sweetener, coating, binder, filler, compression aid,encapsulation aid, preservative, granulation agent, spheronizationagent, stabilizer, adhesive, pigment, sorbent, nanoparticle,microparticle, prodrug, or gel.

In an embodiment, a modified eukaryotic cell comprises at least oneexogenous cellular component from a genetically maternally relatedsource. In an embodiment, the at least one exogenous cellular componentincludes at least one of exogenous mitochondria, exogenous mitochondrialDNA, DNA of the cell nucleus, or exogenous cell nucleus. In anembodiment, the at least one exogenous cellular component is located inat least one intracellular compartment of the modified eukaryotic cell.In an embodiment, the genetically maternally related source includes agenetically maternally related cell.

In an embodiment, a method includes modifying a eukaryotic cell byproviding at least one exogenous cellular component obtained or derivedfrom a maternally genetically related cell. In an embodiment, theexogenous cellular component includes at least one of exogenousmitochondria, exogenous mitochondrial DNA, DNA of the nucleus, orexogenous cell nucleus. In an embodiment, the exogenous mitochondrialDNA of the maternally genetically related cell includes fewer mutationsthan the endogenous mitochondrial DNA of the eukaryotic cell. In anembodiment, the endogenous mitochondrial DNA of the eukaryotic cellincludes fewer mutations than the exogenous mitochondrial DNA of thematernally genetically related cell.

In an embodiment, the at least one of the exogenous mitochondria,exogenous mitochondrial DNA, or exogenous cell nucleus, is provided toat least one intracellular compartment of the eukaryotic cell. In anembodiment, the at least one exogenous cellular component is provided tothe eukaryotic cell by way of at least one of endocytosis, injection orelectroporation. In an embodiment, the intracellular compartment of theeukaryotic cell includes at least one of a nucleolus, cell nucleus,ribosome, vesicle, rough endoplasmic reticulum, Golgi apparatus,cytoskeleton, smooth endoplasmic reticulum, mitochondria, mitochondrialDNA, vacuole, cytoplasm, lysosome, cell wall, vacuole, plastid,chloroplast, leucoplast, chromoplast, ribosome, chromatin, or centriole.

In an embodiment, the method further comprises measuring the membranepotential of the eukaryotic cell or the maternally genetically relatedcell at least one of prior to, during, or subsequent to providing the atleast one exogenous cellular component to the eukaryotic cell. In anembodiment, the at least one exogenous cellular component is extractedfrom the maternally genetically related cell. In an embodiment, themethod further comprises measuring the membrane potential of at leastone of the eukaryotic cell or the maternally genetically related cell atleast one of prior to, during, or subsequent to providing the at leastone exogenous cellular component to the eukaryotic ell.

In an embodiment, at least one exogenous cellular component is providedin response to the presence or level of at least one eukaryotic cellindicator. In an embodiment, the at least one eukaryotic cell indicatorincludes at least one indicator of one or more of a property of theeukaryotic cell; a property of administering the at least onemitochondria, mitochondrial DNA, or cell nucleus, to the eukaryoticcell; eukaryotic cell death; eukaryotic cell division; eukaryotic cellcytoskeletal rearrangement; eukaryotic cell mitochondrial quality,quantity, or arrangement; or eukaryotic cell or tissue secretion.

As described herein, in an embodiment, the property of the eukaryoticcell includes at least one of eukaryotic cell size, eukaryotic cellstage, eukaryotic cell quality, health of the subject from which theeukaryotic cell originates, species of the subject from which theeukaryotic cell originates, immunological background of the eukaryoticcell, mitotic rate of the eukaryotic cell, metabolic profile of theeukaryotic cell, genomic profile of the eukaryotic cell, transcriptomicprofile of the eukaryotic cell, or proteomic profile of the eukaryoticcell, or storage conditions of the eukaryotic cell. In an embodiment,the storage conditions of the eukaryotic cell include at least one ofduration of storage time, storage temperature, storage size, eukaryoticcell dilution, or storage solution(s).

In an embodiment, the at least one eukaryotic cell is selected formodifying based on one or more eukaryotic cell indicators. In anembodiment, the at least one maternally genetically related cell isselected for extraction of the at least one cellular component at leastpartially based on one or more eukaryotic cell indicators. In anembodiment, at least one of the eukaryotic cell or the maternallygenetically related cell includes at least one of a blood cell, musclecell, nerve cell, fibroblast, adipose cell, stem cell, pluripotent cell,epithelial cell, skin cell, liver cell, spleen cell, oocyte, Sertolicell, neoplastic cell, hematopoietic stem cell, lymphocyte, thymocyte,neuronal stem cell, sperm cell, retinal cell, zygote, pancreatic cell,osteoclast, osteoblast, myocyte, embryonic stem cell, fetal cell,embryonic cell, keratinocyte, mucosal cell, mesenchymal stem cell, Tcell, B cell, memory T cell, memory B cell, antigen presenting cell,lymphocyte, thymocyte, meristematic cell, parenchyma cell, collenchymascell, sclerenchyma cell, or other cell.

In an embodiment, the method further comprises selecting the eukaryoticcell for further manipulation. In an embodiment, manipulation includesutilizing the selected eukaryotic cell for transplant or implant into arecipient subject. In an embodiment, manipulation includes at least oneof cell membrane stripping, genetic modification, freezing, or fusingwith another biological cell. In an embodiment, the maternallygenetically related cell is derived from at least one of the mother,biological sibling, sister's child, or mother's sister's child of thesource of the eukaryotic cell. In an embodiment, the maternallygenetically related cell includes or is derived from at least one of anoocyte, or stem cell from at least one of the mother, biologicalsibling, sister's child, or mother's sister's child of the source of theeukaryotic cell. In an embodiment, the maternally genetically relatedcell is derived from the child of the source of the eukaryotic cell,wherein the source of the eukaryotic cell is female. In an embodiment,the maternally genetically related cell includes or is derived from atleast one of an oocyte, or stem cell from the child of the source of theeukaryotic cell. In an embodiment, the source of the exogenous cellularcomponent is of chronologically younger age than the source of theeukaryotic cell. In an embodiment, the source of the exogenous cellularcomponent is of chronologically older age than the source of theeukaryotic cell.

In an embodiment, a method comprises administering a modified eukaryoticcell to a biological tissue; wherein the modified eukaryotic cellincludes at least one exogenous cellular component derived from agenetically maternally related cell. In an embodiment, the at least onebiological tissue is located in a subject. In an embodiment, the atleast one eukaryotic cell is formulated for administration to at leastone biological tissue by at least one route, including, among others,peroral, topical, transdermal, epidermal, intravenous, intraocular,tracheal, transmucosal, intracavity, subcutaneous, intramuscular,inhalation, fetal, intrauterine, intragastric, placental, intranasal,interdermal, intradermal, enteral, parenteral, surgical, or injection.

In an embodiment, the at least one exogenous cellular component includesat least one of exogenous mitochondria, exogenous mitochondrial DNA, DNAof a cell nucleus, or exogenous cell nucleus. In an embodiment, theexogenous mitochondrial DNA of the maternally genetically related cellincludes fewer mutations than the endogenous mitochondrial DNA of theeukaryotic cell. In an embodiment, the endogenous mitochondrial DNA ofthe eukaryotic cell includes fewer mutations than the exogenousmitochondrial DNA of the maternally genetically related cell.

In an embodiment, a method, comprising: selecting at least one exogenouscellular component, at least partially based on one or more geneticcharacteristics of the endogenous mitochondrial DNA of a eukaryoticcell, and providing the at least one selected exogenous cell nucleuscomponent to the eukaryotic cell. In an embodiment, the at least oneexogenous cell nucleus component includes at least one of DNA of thecell nucleus, or an exogenous cell nucleus. In an embodiment, the methodfurther comprises providing at least one of exogenous mitochondria, orexogenous mitochondrial DNA. In an embodiment, the mitochondrial DNAincludes one or more mitochondrial chromosomes. In an embodiment, the atleast one exogenous cell nucleus component is derived from a maternallygenetically related cell. In an embodiment, selecting the at least oneof exogenous cellular component is at least partially based on one ormore alleles of one or more genes of at least one of the eukaryoticcell, or the maternally genetically related cell. In an embodiment, themethod further comprises selecting the at least one exogenous cellularcomponent at least partially based on the Major Histocompatibilitygenetic characteristics of at least one of the source of the eukaryoticcell or the source of the maternally genetically related cell.

In an embodiment, the method further comprises selecting the at leastone exogenous cellular component at least partially based on one or moregenetic characteristics of the DNA of the nucleus of at least one of theeukaryotic cell, or the maternally genetically related cell. In anembodiment, the method further comprises selecting the at least oneexogenous cellular component at least partially based on thecompatibility of the mitochondrial DNA of at least one of the eukaryoticcell, or the maternally genetically related cell. In an embodiment, themethod further comprises selecting the at least one exogenous cellularcomponent at least partially based on the compatibility of one or morealleles of the DNA of the nucleus of the maternally genetically relatedcell with the eukaryotic cell. In an embodiment, selecting the at leastone exogenous cellular component includes searching at least onedatabase including information related to genotyped mitochondrial DNA.

In an embodiment, the method further comprises selecting the at leastone exogenous cellular component at least partially based on thechronological age of at least one of the source of the eukaryotic cell,or the source of the maternally genetically related cell. In anembodiment, the method further comprises selecting the at least oneexogenous cellular component at least partially based on the number ofmutations in the mitochondrial DNA of at least one of the eukaryoticcell, or the maternally genetically related cell.

In an embodiment, the at least one exogenous cellular component isprovided to at least one intracellular compartment of the eukaryoticcell. As described herein, in an embodiment, the at least oneintracellular compartment includes at least one of a nucleolus, nucleus,ribosome, vesicle, rough endoplasmic reticulum, Golgi apparatus,cytoskeleton, smooth endoplasmic reticulum, mitochondria, vacuole,cytoplasm, lysosome, or centriole. In an embodiment, at least oneendogenous mitochondria, mitochondrial DNA, cell nucleus, ornucleocytoplasm, is at least partially removed from the eukaryotic cellprior to providing the at least one exogenous cellular component to theeukaryotic cell. In an embodiment, at least one of the endogenousmitochondria, mitochondrial DNA, cell nucleus, or nucleocytoplasm, isremoved beyond detection from the eukaryotic cell prior to providing theat least one exogenous cellular component to the eukaryotic cell. In anembodiment, the method further comprises selecting the at least oneexogenous cellular component at least partially based on the MajorHistocompatibility type of at least one of the source of the eukaryoticcell or the maternally genetically related cell.

In an embodiment, the detection material includes at least one of aradioactive, luminescent, colorimetric fluorescent or odorous substance.In an embodiment, the at least one detection material includes at leastone of a taggant, contrast agent, sensor, or electronic identificationdevice. In an embodiment, the at least one electronic identificationdevice includes at least one radio frequency identification device. Inan embodiment, the at least one sensor receives information associatedwith at least one of temperature, pH, inflammation, presence of at leastone substance, or biological response to administration of thecomposition. In an embodiment, the at least one detection materialincludes at least one of a diamagnetic particle, ferromagnetic particle,paramagnetic particle, super paramagnetic particle, particle withaltered isotope, or other magnetic particle.

In an embodiment, one or more SNPs may alter one or more of a codingregion, gene product, non-coding region, intergenic region, centromericregion, telomeric region, or RNA. In an embodiment, the one or more SNPsmay be in linkage disequilibrium with one or more traits, alleles, ormarkers of chromosomal characteristics.

In an embodiment, one or more chromosomal characteristics include, amongother things, one or more duplications, insertions, deletions,substitutions, replications, or breaks. In an embodiment, one or morechromosomal characteristics include haplotype or nucleic acid sequence.In an embodiment, one or more nucleic acid sequences include at leastone of a repetitive sequence, telomeric sequence, centromeric sequence,mutated sequence, alternate sequence, intergenic sequence, proteincoding sequence, or non-coding sequence. In an embodiment, the nucleicacid sequence is linked with one or more of a disease, disorder,syndrome, or condition, and optionally may encode a gene linked with oneor more of a disease, disorder, syndrome, or condition.

In an embodiment, one or more genetic characteristics include one ormore of a single nucleotide polymorphism, chromosomal characteristic,methylation pattern, or nucleic acid sequence. In an embodiment, one ormore of these or other genetic characteristics are detected in at leastone of the donor cell or recipient cell. In an embodiment, one or moreof these genetic characteristics are utilized in analysis of at leastone of the donor cell or recipient cell.

In an embodiment, at least one of the endogenous mitochondria,mitochondrial DNA, or cell nucleus is at least partially removed fromthe eukaryotic cell prior to providing at least one of the exogenousmitochondria, mitochondrial DNA, or cell nucleus. In an embodiment, theendogenous mitochondria, mitochondrial DNA, or cell nucleus is at leastabout 99%, at least about 95%, at least about 90%, at least about 85%,at least about 80%, at least about 75%, at least about 70%, at leastabout 65%, at least about 60%, at least about 55%, at least about 50%,at least about 45%, at least about 40%, at least about 35%, at leastabout 30%, at least about 25%, at least about 20%, at least about 15%,at least about 10%, at least about 5%, at least about 1%, or any valueless than or therebetween.

In an embodiment, a modified eukaryotic cell, produced by the process ofproviding at least one exogenous cellular component selected at leastpartially based on one or more genetic characteristics of the endogenousmitochondrial DNA. In an embodiment, the at least one exogenous cellularcomponent includes at least one of exogenous mitochondria, exogenousmitochondrial DNA, or exogenous cell nucleus.

In an embodiment, a modified eukaryotic cell comprises at least oneexogenous cellular component compatible with the genetic characteristicsof the endogenous mitochondrial DNA of the eukaryotic cell.

Kits

In an embodiment, kits are included for any of the various aspectsdisclosed herein. For example, in an embodiment, a kit includes adetection material responsive to at least one eukaryotic cell indicator,and means for administering at least one exogenous energy supplyingfactor to at least one eukaryotic cell. In an embodiment, the kitincludes the at least one exogenous energy supplying factor (e.g.,pyruvate, ATP, glucose or other carbohydrate, etc.). In an embodiment,the kit includes a delivery device. In an embodiment, the kit includesat least one tool for selecting at least one eukaryotic cell formanipulation. In an embodiment, a kit includes standard packaging orinstructions for use.

In an embodiment, a kit comprises: a detection material responsive to atleast one eukayrotic cell indicator, and means for providing at leastone exogenous cellular component to at least one eukaryotic cell. In anembodiment, the detection material includes at least one of aradioactive, luminescent, colorimetric fluorescent, or odoroussubstance. In an embodiment, the at least one detection materialincludes at least one of a taggant, contrast agent, magnetic particle,particle with altered isotope, or electronic identification device. Inan embodiment, the at least one electronic identification deviceincludes at least one radio frequency identification device. In anembodiment, the at least one magnetic particle includes at least oneparamagnetic particle, ferromagnetic particle, super paramagneticparticle, diamagnetic particle, or other magnetic particle. In anembodiment, the kit further comprises at least one sensor. In anembodiment, the at least one sensor is configured to receive informationassociated with at least one of temperature, pH, inflammation, presenceof at least one substance, detection material, or biological response toadministration of at least one of the exogenous mitochondria,mitochondrial DNA, cell nucleus, or nucleocytoplasm. In an embodiment,the at least one sensor is configured to receive information associatedwith at least one eukaryotic cell indicator. In an embodiment, the atleast one detection material includes at least one of a diamagneticparticle, ferromagnetic particle, paramagnetic particle, superparamagnetic particle, particle with altered isotope, or other magneticparticle.

In an embodiment, a kit comprises: a detection material responsive to atleast one eukayrotic cell indicator, and means for providing at leastone exogenous cellular component to at least one eukaryotic cell,wherein the exogenous cellular component is selected based at least inpart on one or more genetic characteristics of the endogenousmitochondrial DNA. In an embodiment, the detection material includes atleast one of a radioactive, luminescent, colorimetric fluorescent, orodorous substance. In an embodiment, the at least one detection materialincludes at least one of a taggant, contrast agent, magnetic particle,particle with altered isotope, or electronic identification device. Inan embodiment, the at least one electronic identification deviceincludes at least one radio frequency identification device.

In an embodiment, the at least one magnetic particle includes at leastone paramagnetic particle, ferromagnetic particle, super paramagneticparticle, diamagnetic particle, or other magnetic particle. In anembodiment, the kit further comprises at least one sensor. In anembodiment, the at least one sensor is configured to receive informationassociated with at least one of temperature, pH, inflammation, presenceof at least one substance, detection material, or biological response toadministration of at least one of the exogenous mitochondria,mitochondrial DNA, cell nucleus, or nucleocytoplasm. In an embodiment,the at least one sensor is configured to receive information associatedwith at least one eukaryotic cell indicator. In an embodiment, the atleast one detection material includes at least one of a diamagneticparticle, ferromagnetic particle, paramagnetic particle, superparamagnetic particle, particle with altered isotope, or other magneticparticle.

As indicated in the Figures, FIG. 1 illustrates a cell 100, including acell membrane 130, a nucleus 120 with a membrane of the nucleus 140,cytoplasm 160, endoplasmic reticulum 170, Golgi bodies 180, mitochondria150. In an embodiment, at least one mitochondrion of the donor cell istransplanted to the recipient cell. In an embodiment, the donor andrecipient cells are maternally genetically related.

FIG. 2 illustrates a transverse view of an example of a mitochondrion200, including cristae 220, mitochondrial DNA 230, granules 210, anouter mitochondrial membrane 260, an inter membrane space 240, an innermatrix 270, and an inner mitochondrial membrane 250.

FIG. 3 illustrates a method, comprising 310 receiving on acomputer-readable medium, a first input associated with a first possibledataset, the first possible dataset including data representative of oneor more mitochondrial DNA characteristics; and determining one or moreparameters for selecting at least one exogenous cellular component fortransfer to a eukaryotic cell, based on the first possible dataset. Inan embodiment 320, the one or more mitochondrial DNA characteristicsinclude one or more endogenous mitochondrial DNA characteristics. In anembodiment 330, the one or more mitochondrial DNA characteristicsinclude one or more exogenous mitochondrial DNA characteristics from aeukaryotic donor cell. In an embodiment 340, the one or moremitochondrial DNA characteristics include one or more exogenousmitochondrial DNA characteristics from a maternally genetically relatedcell. In an embodiment, 350 the method further comprises a secondpossible dataset including data representative of one or more cellularcharacteristics of the eukaryotic cell. In an embodiment, 360 the methodfurther comprises a second possible dataset including datarepresentative of one or more cellular characteristics of a donor cell.In an embodiment 370, the donor cell includes a maternally geneticallyrelated cell. In an embodiment 380, the method further comprises asecond possible dataset including data representative of one or moremitochondrial DNA characteristics of the eukaryotic cell. In anembodiment 390, the method further comprises a second possible datasetincluding data representative of one or more mitochondrial DNAcharacteristics of a donor cell. In an embodiment 392, the donor cellincludes a maternally genetically related cell. In an embodiment 394,the method further comprises a second possible dataset including datarepresentative of at least one actual or predicted interaction between anucleus DNA-derived protein with a mitochondrial DNA-derived protein.

As illustrated in FIG. 4, in an embodiment 400, the at least oneexogenous cellular component includes at least one of exogenousmitochondria, exogenous mitochondrial DNA, exogenous cell nucleus,exogenous DNA from the nucleus, or exogenous nucleocytoplasm. In anembodiment 410, wherein receiving on a computer-readable medium a firstinput associated with a first possible dataset, comprises: receiving thefirst input associated with the first possible dataset, the first inputincluding data representative of one or more of the one or moremitochondrial DNA characteristics. In an embodiment 420, whereinreceiving on a computer-readable medium a first input associated with afirst possible dataset, comprises: receiving the first input associatedwith the first possible dataset, the first input including datarepresentative of one or more of the one or more mitochondrial DNAcharacteristics, including at least one of a genetic attribute, singlenucleotide polymorphism, haplotype, allelic marker, allele, diseasemarker, genetic abnormality, genetic disease, genetic mutation,inversion, deletion, duplication, recombination, nucleic acid sequence,gene, protein coding sequence, intron, exon, regulatory sequence,intergenic sequence, mitochondrial nucleic acid sequence, mitochondria,methylation pattern, or epigenetic element.

In an embodiment 430, wherein receiving on a computer-readable medium afirst input associated with a first possible dataset comprises:receiving the first input associated with the first possible dataset,the first input including data representative of one or moremitochondrial DNA characteristics of at least one of a genome, ornucleic acid. In an embodiment 440, receiving on a computer-readablemedium a first input associated with a first possible dataset comprises:receiving a first data entry associated with the first possible dataset.In an embodiment 450, receiving on a computer-readable medium a firstinput associated with a first possible dataset comprises: receiving afirst data entry associated with the first possible dataset, the firstdata entry including data representative of one or more of the one ormore mitochondrial DNA characteristics. In an embodiment 460, whereinreceiving on a computer-readable medium a first data entry associatedwith a first possible dataset, the first data entry including datarepresentative of one or more of the at least one mitochondrial DNAcharacteristics, comprises: receiving on a computer-readable medium afirst data entry associated with the first possible dataset, the firstdata entry including data representative of one or more of the one ormore mitochondrial DNA characteristics, including at least one of agenetic attribute, single nucleotide polymorphism, haplotype, allelicmarker, allele, disease marker, genetic abnormality, genetic disease,chromosomal abnormality, genetic mutation, inversion, deletion,duplication, recombination, chromosome, nucleic acid sequence, gene,protein coding sequence, intron, exon, regulatory sequence, intergenicsequence, mitochondrial nucleic acid sequence, mitochondria, telomere,telomere repeat, telomere length, centromere repeat, centromere,methylation pattern, or epigenetic element.

As illustrated in FIG. 5, in an embodiment 500, receiving on acomputer-readable medium a first input associated with a first possibledataset comprises: receiving on a computer-readable medium a first dataentry from a graphical user interface. In an embodiment 510, receivingon a computer-readable medium a first input associated with a firstpossible dataset comprises: receiving on a computer-readable medium afirst data entry from at least one submission element of a graphicaluser interface. In an embodiment 520, receiving on a computer-readablemedium a first input associated with a first possible dataset comprises:receiving on a computer-readable medium a first data entry at leastpartially identifying one or more elements of the first possibledataset. In an embodiment 530, receiving on a computer-readable medium afirst data entry at least partially identifying one or more elements ofthe first possible dataset comprises: receiving on a computer-readablemedium the first data entry at least partially identifying the one ormore elements of the first possible dataset, one or more of the one ormore elements including data representative of one or more geneticcharacteristics. In an embodiment 540, receiving on a computer-readablemedium a first data entry at least partially identifying the one or moreelements of the first possible dataset comprises: receiving on acomputer-readable medium the first data entry at least partiallyidentifying one or more elements of the first possible dataset, one ormore of the one or more elements including data representative of one ormore of the at least one of a genome, chromosome, or nucleic acid. In anembodiment 550, receiving on a computer-readable medium a first dataentry at least partially identifying one or more elements of the firstpossible dataset comprises: receiving on a computer-readable medium thefirst data entry at least partially identifying the one or more elementsof the first possible dataset, one or more of the one or more elementsincluding data representative of at least one biological tissue orbiological cell. In an embodiment 560, the at least one biological cellincludes one of a blood cell, muscle cell, nerve cell, fibroblast,adipose cell, stem cell, pluripotent cell, epithelial cell, skin cell,liver cell, spleen cell, oocyte, Sertoli cell, neoplastic cell,hematopoietic stem cell, lymphocyte, thymocyte, neuronal stem cell,sperm cell, retinal cell, pancreatic cell, osteoclast, osteoblast,myocyte, embryonic stem cell, fetal cell, embryonic cell, keratinocyte,mucosal cell, mesenchymal stem cell, T cell, B cell, memory T cell,memory B cell, antigen presenting cell, lymphocyte, thymocyte,meristematic cell, parenchyma cell, collenchymas cell, sclerenchymacell, or other cell.

As illustrated in FIG. 6, in an embodiment 600, the method furthercomprises accessing the first possible dataset in response to the firstinput. In an embodiment 610, accessing the first possible dataset inresponse to the first input comprises: accessing the first possibledataset in response to the first input, the first input including datarepresentative of one or more of the at least one mitochondrial geneticcharacteristic. In an embodiment 620, accessing the first possibledataset in response to the first input comprises: accessing the firstpossible dataset from within a first database associated with aplurality of genetic characteristics. In an embodiment 630, accessingthe first possible dataset in response to the first input comprises:accessing the first possible dataset by associating one or more of theat least one mitochondrial genetic characteristic with one or moreelements of the first possible dataset.

In an embodiment 640, accessing the first possible dataset in responseto the first input comprises: accessing the first possible dataset usinga database management system engine configured to query a first databaseto retrieve the first possible dataset therefrom. In an embodiment 650,accessing the first possible dataset in response to the first inputcomprises: accessing the first possible dataset by corresponding one ormore of the at least one mitochondrial genetic characteristic with oneor more elements of the first possible dataset. In an embodiment 660,accessing the first possible dataset by corresponding one or more of theone or more mitochondrial DNA characteristics with one or more elementsof the first possible dataset comprises: accessing the first possibledataset by corresponding one or more of the one or more mitochondrialDNA characteristics including at least one of a genetic attribute,single nucleotide polymorphism, haplotype, allelic marker, allele,disease marker, genetic abnormality, genetic disease, chromosomalabnormality, genetic mutation, inversion, deletion, duplication,recombination, chromosome, nucleic acid sequence, gene, protein codingsequence, intron, exon, regulatory sequence, intergenic sequence,mitochondrial nucleic acid sequence, mitochondria, telomere, telomererepeat, telomere length, centromere repeat, centromere, methylationpattern, or epigenetic element with the one or more elements of thefirst possible dataset.

As illustrated in FIG. 7, in an embodiment 700, accessing the firstpossible dataset in response to the first input comprises: accessing thefirst possible dataset as being associated with one or more of the oneor more mitochondrial DNA characteristics, based on one or morecharacterizations stored in association with one or more elements of thefirst possible dataset. In an embodiment 710, accessing the firstpossible dataset in response to the first input comprises: accessing thefirst possible dataset as being associated with one or more of the oneor more mitochondrial DNA characteristics, based on one or morecharacterizations stored in association with one or more elements of thefirst possible dataset, the one or more elements including one or moregenetic characteristics. In an embodiment 720, receiving on acomputer-readable medium a first input associated with a first possibledataset comprises: receiving on a computer-readable medium a firstrequest associated with the first possible dataset. In an embodiment730, receiving on a computer-readable medium a first input associatedwith a first possible dataset comprises: receiving on acomputer-readable medium a first request associated with the firstpossible dataset, the first request selecting one or more of the one ormore mitochondrial DNA characteristics.

In an embodiment 740, receiving a first input associated with a firstpossible dataset comprises: receiving on a computer-readable medium afirst request from a graphical user interface. In an embodiment 750,receiving on a computer-readable medium a first input associated with afirst possible dataset comprises: receiving on a computer-readablemedium a first request from at least one submission element of agraphical user interface. In an embodiment 760, receiving on acomputer-readable medium a first input associated with a first possibledataset comprises: receiving on a computer readable medium a firstrequest, the first request at least partially identifying one or moreelements of the first possible dataset. In an embodiment 770, receivingon a computer-readable medium a first input associated with a firstpossible dataset comprises: receiving on a computer-readable medium afirst request, the first request selecting one or more elements of thefirst possible dataset. In an embodiment 780, receiving on acomputer-readable medium a first input associated with a first possibledataset comprises: receiving on a computer-readable medium a firstrequest, the first request providing instructions at least partiallyidentifying one or more of the one or more mitochondrial DNAcharacteristics.

As illustrated in FIG. 8, in an embodiment 800, receiving on acomputer-readable medium a first input associated with a first possibledataset comprises: receiving on a computer-readable medium a firstrequest, the first request providing instructions for determining one ormore of the one or more mitochondrial DNA characteristics. In anembodiment 810, receiving on a computer-readable medium a first inputassociated with a first possible dataset comprises: accessing the firstpossible dataset in response to a first request, the first requestspecifying one or more of the one or more mitochondrial DNAcharacteristics and at least one other instruction. In an embodiment820, the method further comprises: generating with a computer-recordablemedium, the first possible dataset in response to the first input. In anembodiment 830, generating with a computer-recordable medium, the firstpossible dataset in response to the first input comprises: generatingwith a computer-recordable medium, the first possible dataset inresponse to the first input, the first input including datarepresentative of one or more of the one or more mitochondrial DNAcharacteristics. In an embodiment 840, generating with acomputer-recordable medium the first possible dataset in response to thefirst input comprises: generating with a computer-recordable medium, thefirst possible dataset from within a first database associated with aplurality of genetic characteristics.

In an embodiment 850, generating with a computer-recordable medium, thefirst possible dataset in response to the first input comprises:generating with a computer-recordable medium, the first possible datasetby associating one or more of the one or more mitochondrial DNAcharacteristics with one or more elements of the first possible dataset.In an embodiment 860, generating with a computer-recordable medium, thefirst possible dataset in response to the first input comprises:generating with a computer-recordable medium the first possible datasetusing a database management system engine configured to query a firstdatabase to retrieve the first possible dataset therefrom.

As illustrated in FIG. 9, in an embodiment 900, generating with acomputer-recordable medium, the first possible dataset in response tothe first input comprises: generating with a computer-recordable medium,the first possible dataset by corresponding one or more of the one ormore mitochondrial DNA characteristics with one or more elements of thefirst possible dataset. In an embodiment 910, receiving on acomputer-readable medium a first input associated with a first possibledataset comprises: receiving on a computer-readable medium a firstrequest associated with the first possible dataset. In an embodiment920, receiving on a computer-readable medium the first input associatedwith a first possible dataset comprises: receiving on acomputer-readable medium a first request associated with the firstpossible dataset, the first request selecting one or more of the one ormore mitochondrial DNA characteristics. In an embodiment 930, receivingon a computer-readable medium a first input associated with a firstpossible dataset comprises: receiving on a computer-readable medium afirst request from a graphical user interface. In an embodiment 930,receiving on a computer-readable medium a first input associated with afirst possible dataset comprises: receiving on a computer-readablemedium a first request from at least one submission element of agraphical user interface. In an embodiment 940, receiving on acomputer-readable medium a first input associated with a first possibledataset comprises: receiving on a computer-readable medium a firstrequest, the first request at least partially identifying one or moreelements of the first possible dataset. In an embodiment 950, receivingon a computer-readable medium a first input associated with a firstpossible dataset comprises: receiving on a computer-readable medium afirst request, the first request selecting one or more elements of thefirst possible dataset. In an embodiment 960, receiving on acomputer-readable medium a first input associated with a first possibledataset comprises: receiving on a computer-readable medium a firstrequest, the first request providing instructions at least partiallyidentifying one or more of the one or more mitochondrial DNAcharacteristics. In an embodiment 970, receiving on a computer-readablemedium a first input associated with a first possible dataset comprises:receiving on a computer-readable medium a first request, the firstrequest providing instructions for determining one or more of the one ormore mitochondrial characteristics.

As illustrated in FIG. 10, in an embodiment 1000, receiving on acomputer-readable medium a first input associated with a first possibledataset comprises: receiving on a computer-readable medium a firstrequest associated with the first possible dataset; and generating witha computer-recordable medium the first possible dataset in response tothe first request, the first request specifying one or more of the oneor more mitochondrial DNA characteristics and at least one otherinstruction. In an embodiment 1010, receiving on a computer-readablemedium a first input associated with a first possible dataset comprises:receiving on a computer-readable medium a first request, the firstrequest specifying one or more of the one or more mitochondrial DNAcharacteristics; and generating with a computer-recordable medium thefirst possible dataset in response to the first request at leastpartially by performing an analysis of one or more of the one or moremitochondrial DNA characteristics. In an embodiment 1020, the methodfurther comprises determining a graphical illustration of the firstpossible dataset. In an embodiment 1030, determining a graphicalillustration of the first possible dataset comprises: determining thegraphical illustration for inclusion in a display element of a graphicaluser interface. In an embodiment 1040, determining a graphicalillustration of the first possible dataset comprises: performing ananalysis of one or more elements of the first possible dataset todetermine a first possible outcome; and determining the graphicalillustration based at least in part on the analysis. In an embodiment1050, determining a graphical illustration of the first possible datasetcomprises: performing an analysis of one or more elements of the firstpossible dataset to determine a first possible outcome, the firstpossible outcome including one or more of a possible risk, a possibleresult, a possible consequence, a likelihood of success, or a cost; anddetermining the graphical illustration based on the analysis. In anembodiment 1060, determining a graphical illustration of the possibledataset comprises: performing an analysis of one or more elements of thefirst possible dataset to determine a first possible outcome, the firstpossible outcome including one or more of a predicted risk, predictedresult, predicted consequence; and determining the graphicalillustration based on the analysis. In an embodiment 1070, determiningthe graphical illustration of the first possible dataset comprises:performing an analysis of one or more elements of the first possibledataset to determine a first possible outcome, the first possibleoutcome including one or more of a predicted risk, predicted result, orpredicted consequence; and determining the graphical illustrationincluding one or more of the one or more mitochondrial characteristicsin association with a visual indicator related to the first possibleoutcome.

As illustrated in FIG. 11, in an embodiment 1100, determining agraphical illustration of the first possible dataset comprises:determining a correlation between a first possible outcome and a type orcharacteristic of a visual indicator used in the graphical illustrationto represent the first possible outcome. In an embodiment 1110, the atleast one exogenous cellular component is derived from a maternallygenetically related cell.

As illustrated in FIG. 12, in an embodiment, a computer program product,comprises 1210 a computer-recordable medium bearing at least one of oneor more instructions for receiving a first input associated with a firstpossible dataset, the first possible dataset including datarepresentative of one or more mitochondrial DNA characteristics; whereinat least one of the one or more mitochondrial DNA characteristics is agenetic characteristic; and one or more instructions for determiningparameters for selecting one or more characteristics based on one ormore of a mitochondria, mitochondrial DNA, cell nucleus, ornucleocytoplasm, from the first possible dataset. In an embodiment 1220,the computer program product further comprises one or more instructionsfor accessing the first possible dataset in response to the first input.In an embodiment 1230, the computer program product further comprisesone or more instructions for generating the first possible dataset inresponse to the first input. In an embodiment 1240, the computer programproduct further comprises: one or more instructions for determining agraphical illustration of the first possible dataset. In an embodiment1250, the computer-recordable medium includes a computer-readablemedium. In an embodiment 1260, the computer-recordable medium includes arecordable medium. In an embodiment 1270, the computer-recordable mediumincludes a communications medium.

As illustrated in FIG. 13, in an embodiment 1300, a system comprises1310 a computing device; and instructions that when executed on thecomputing device cause the computing device to receive a first inputassociated with a first possible dataset, the first possible datasetincluding data representative of one or more mitochondrial DNAcharacteristics; wherein at least one of the one or more mitochondrialDNA characteristics is a genetic characteristic; and instructions thatwhen executed on the computing device cause the computing device todetermine parameters for selecting at least one exogenous cellularcomponent for transfer to a eukaryotic cell based on the first possibledataset. In an embodiment 1320, the at least one exogenous cellularcomponent includes at least one of exogenous mitochondria, exogenousmitochondrial DNA, exogenous cell nucleus, or exogenous nucleocytoplasm.In an embodiment 1330, the at least one exogenous cellular component isderived from a maternally genetically related cell. In an embodiment1340, the system further comprises: instructions that when executed onthe computing device cause the computing device to access the firstpossible dataset in response to the first input. In an embodiment 1350,the system further comprises: instructions that when executed on thecomputing device cause the computing device to generate the firstpossible dataset in response to the first input. In an embodiment, thesystem further comprises: instructions that when executed on thecomputing device cause the computing device to determine a graphicalillustration of the first possible dataset. In an embodiment 1360, thecomputing device comprises: one or more of a desktop computer,workstation computer, computing system, cluster of processors, networkedcomputer, tablet personal computer, laptop computer, or personal digitalassistant. In an embodiment 1370, the computing device is operable tocommunicate with a database to access the first possible dataset. In anembodiment 1380, the computing device is operable to communicate with anapparatus configured to select the at least one exogenous cellularcomponent for transfer into the eukaryotic cell.

The following Examples are intended to be illustrative of particularembodiments, and are not intended to be limiting in any way.

PROPHETIC EXAMPLES Example 1 Modification of Mesenchymal Stem Cells withMaternally Genetically Related Mitochondria

A method is described for modifying mesenchymal stem cells withmitochondria from maternally genetically related donor cells prior toautologous transplantation of the stem cells into a subject withamyotrophic lateral sclerosis (ALS). Autologous mesenchymal stem celltransplantation into the spinal cord is being used as a treatment optionto attenuate neurodegeneration in individuals with ALS (Mazzini, et al.Exp. Neurology; Abstract; 223:229-237 (2010), which is incorporatedherein by reference). Impaired mitochondrial function is a potentialcausal factor in neuronal dysfunction and degeneration in adultneurodegenerative diseases and is exemplified by reduced mitochondrialgene copy numbers and increased mitochondrial DNA gene deletions insurviving spinal neurons of individuals with ALS (Keeney & Bennett, Mol.Neurodegener. 5:21 (2010), which is incorporated herein by reference).Modifying mesenchymal stem cells with mitochondria from healthymaternally genetically related cells prior to autologous transplantationprovides a means for improving mitochondrial function.

Platelets are used as a minimally invasive source of donor mitochondria.In addition, platelets lack DNA of the nucleus and are capable oftransferring mitochondria to target cells through cellular fusion(Bacman & Moraes Methods Cell Biol. 80:503-524 (2007), which isincorporated herein by reference). Blood is drawn from a maternallygenetically related subject into sterile acid citrate dextroseanticoagulant and centrifuged twice at 200×g for 15 min to pellet redand white blood cells; the resultant platelet-rich plasma is used as thesource of platelets. Platelets are isolated by centrifugation of theplatelet-rich plasma at 1,400×g for 10 min, and washed twice in washingsolution 1 (12.72 mM sodium citrate, 2.99 mM glucose, 9.41 mM NaCl, 0.55mM EDTA), and washed once in washing solution 2 (1 mM Tris-HCl, 2.99 mMglucose, 15.38 mM NaCl, 0.55 mM EDTA, pH 7.4).

Mitochondria are isolated from the platelets by cell lysis, followed bydifferential centrifugation (Silvagno, et al. PLoS ONE 5(1):e8670(2010), which is incorporated herein by reference). Briefly, theplatelets are resuspended in a hypotonic lysis buffer (10 mM Tris-HCl,pH 7.4 and 1% protease inhibitor solution) and incubated on ice for 30minutes. The platelets are lysed by five passages through an insulinsyringe needle to generate a total cell extract. The total cell extractis layered on a linear 30-50% sucrose gradient and centrifuged at134,000×g for 120 minutes in a Beckman SW40 Ti rotor. The resultingparticulate bands are diluted in 0.29 M sucrose and pelleted bycentrifugation at 100,000×g for 60 minutes.

Alternatively, mitochondria are isolated from lysed platelets using acommercially available mitochondria isolation kit (e.g. MitochondriaIsolation Kit for Cultured Cells, MitoSciences, Eugene, Oreg.). Thedonor mitochondria isolated from the maternally genetically relatedcells are analyzed for presence or absence of specific mitochondrialproperties prior to insertion of the donor mitochondria into mesenchymalstem cells. Mitochondrial DNA in intact mitochondria can be screened formutations using specific peptide nucleic acid (PNA) oligomers conjugatedto a membrane permeable reagent such as, for example,triphenylphosphonium (see, e.g., Muratovska, et al. Nucleic Acids Res.29:1852-1863 (2001), which is incorporated herein by reference). A PNAoligomer is designed with a DNA sequence complimentary to a specificmitochondrial sequence, either normal or mutated, and used to assesswhether a specific mitochondria contains DNA with that specificsequence.

For this analysis, PNA oligomers are conjugated to triphenylphosphoniumby pretreating the PNA with 10 mM HEPES, 1 mM EDTA, and 250 nM2-mercaptoethanol for 1 hour at 40° C. followed by incubation withiodobutyltriphenylphosphonium in HEPES/EDTA/ethanol for an additional 4hours. The conjugated PNA is purified using reverse phase HPLC. The PNAis further conjugated to a fluorescent dye, such as FITC, TRITC, and/orBODIPY® derivatives, and/or quantum dots (see, e.g., Dahan Histochem.Cell Biol.; Abstract; 125:451-456 (2006), which is incorporated hereinby reference). Relative binding of the fluorescent PNA to thecomplementary site within the mitochondrial DNA is assessed using eitherfluorescence microscopy, or fluorescence activated cell sorting.Additional PNA probes are used as negative and positive controls of DNAbinding to ensure that the assay system is working. The analysis ofdonor mitochondria can be compared with that of the recipientmitochondria and/or published information regarding mitochondrial DNA toensure that the donor mitochondria are healthy.

Autologous mesenchymal stem cells are isolated from the bone marrow ofthe ALS patient. Bone marrow is collected by aseptic aspiration from theposterior iliac crest using standard procedures. Ex vivo expansion ofmesenchymal stem cells is carried out essentially as described byPittenger, et al. Science, Abstract; 284:143-147 (1999), which isincorporated herein by reference). Briefly, the bone marrow is dilutedin four volumes of Minimum Essential Medium Alpha Medium containing 10%fetal bovine serum and centrifuged at 900×g for 15 minutes. The cellsare washed in phosphate-buffered saline and plated into an expansionmedium containing Dulbecco's Minimum Essential Medium supplemented with40% MCDB-201, 10% fetal bovine serum, insulin-transferrin-sodiumselenite supplement, linoleic acid-bovine serum albumin, 10⁻⁸ Mdexamethasone, 10⁻⁴ M ascorbic acid 2-phosphate, 50 U/mlpenicillin/streptomycin, 10 ng/ml human platelet-derived growthfactor-BB, and 10 ng/ml human epithelial growth factor. The cells areexpanded over several passages prior to transplantation.

Donor mitochondria isolated from the maternally genetically relatedcells are inserted into cells with existing endogenous mitochondrialDNA, creating a heteroplasmic state. Alternatively, donor mitochondriaare inserted into cells lacking endogenous mitochondrial DNA. Cellslacking mitochondrial DNA, termed ρ⁰ cells, are generated by sustainedtreatment of cells with a toxin, e.g., ethidium bromide, at aconcentration that blocks mitochondrial DNA replication with onlyminimal effect on DNA of the nucleus (see, e.g., U.S. Pat. No.5,888,498; Yoon & Koob, Nucleic Acids Res. 31:1407-1415 (2003), whichare incorporated herein by reference). Briefly, the mesenchymal stemcells isolated from a patient with ALS are exposed to 5 μg/ml ethidiumbromide for 4 weeks in high-glucose medium supplemented with 50 μg/mluridine and 0.1 mg/ml sodium pyruvate. At the end of 4 weeks, clonal ρ⁰cells are isolated by clonal dilution of the ethidium bromide treatedcells. PCR with mitochondrial DNA specific primers as well as enzymeassays for cyanide-inhibitable oxygen utilization and complex I orcytochrome c oxidase activity are used to verify the ρ⁰ state of aclonal population. ρ⁰ cells can also be generated using othermitochondrial toxins including ditercalinium, rhodamine 6G,dideoxycytidine, streptozotocin (Inoue et al. J. Biol. Chem.272:15510-15515 (1997); Bacman & Moraes, Methods Cell Biol. 80:503-524(2007), which are incorporated herein by reference).

The donor mitochondria from the maternally genetically related cells aretransferred into the mesenchymal stem cells using cellular fusion inwhich a cell containing mitochondrial DNA, e.g., a donor platelet, isfused with a cell lacking mitochondrial DNA, e.g., ρ⁰ mesenchymal stemcells (see, e.g. Bacman & Moraes, Methods Cell Biol. 80:503-524 (2007);Kagawa & Hayashi Gene Ther. 4:6-10 (1997); Pye et al. Nucleic Acids Res.34:e95 (2006) which are incorporated herein by reference). Fusion iscarried out by incubating the two cell populations in the presence ofpolyethylene glycol (PEG). The ρ⁰ mesenchymal stem cells are combinedwith the donor platelets for several hours in growth medium, allowingcell-cell contacts to be made. The medium is completely removed, and PEG1450 is added to the cells for a brief 30 to 60 second period at whichtime the PEG is removed and the cells washed. The cells are culturedunder selection conditions such that only the fused cells survive.

The modified mesenchymal stem cells are suspended in 1-2 ml ofautologous cerebrospinal fluid and injected at multiple sites using amicrometric pump injector into the anterior horns of spinal cord of theALS patient.

Example 2 Modification of Myoblast Cells with Maternally GeneticallyRelated Mitochondrial DNA

A method is described for modifying myoblast cells with mitochondriafrom maternally genetically related donor cells prior to autologoustransplantation of the myoblast cells into a subject with heart failure.Autologous myoblast cell transplantation into cardiac tissue has beentested as a treatment option for individuals with congestive heartfailure and myocardial infarction, as a means for replacing irreversiblydamaged cardiomyocytes (see, e.g., Opie and Dib, Nat. Clin. Pract.Cardiovasc. Med. 3 Suppl 1:S42-5 (2006), which is incorporated herein byreference). Impaired mitochondrial function associated with aging is apotential causal factor in the decline in cardiac muscle function (see,e.g., Marin-Garcia, et al. Cardiovasc. Drugs Ther.; Abstract; 20:477-491(2006), which is incorporated herein by reference). Modifying myoblastcells with mitochondria from healthy maternally genetically relatedcells prior to autologous transplantation provides a means for improvingmitochondrial function.

Lymphocytes are used as a minimally invasive source of donormitochondria (Choo-Kang et al. Diabetes 51:2317-2320 (2002), which isincorporated herein by reference). Blood is drawn from a maternallygenetically related subject using standard procedures. Ideally the donoris younger than the intended recipient. Lymphocytes are isolated fromthe blood by centrifugation through Ficoll-Hypaque Plus (AmershamBiosciences, Piscataway, N.J.) and incubated for 3 hours at 37° C. inRPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 50units/ml penicillin and 50 mg/ml streptomycin. After 3 hours, thenon-adherent lymphocytes are removed and further cultured in freshmedium for an additional 48 hours.

Mitochondria are isolated from the lymphocytes by cell lysis, followedby differential centrifugation (Carpentieri & Sordahl, Cancer Res.40:221-224 (1980), which is incorporated herein by reference). Briefly,the lymphocytes are lysed in 0.25 M sucrose, 5 mM Tri-HCl, 5 mM EGTA,and 0.5% bovine serum albumin using a tight fitting Teflon® pestleattached to a motor rotating at approximately 6000 rpm, a tissuehomogenizer (e.g. Tekmar Tissumizer, Tekmar Co. Cincinnati Ohio), or aDounce homogenizer. The homogenate is centrifuged at 480×g for 10minutes to remove nuclei and heavier cellular debris. The supernatant isthen centrifuged at 10,000×g for 10 minutes to generate a mitochondrialpellet. Alternatively, mitochondria are isolated from lymphocytes usinga commercially available mitochondria isolation kit (e.g. MitochondriaIsolation Kit for Cultured Cells, MitoSciences, Eugene, Oreg.).

To isolate mitochondrial DNA, the isolated mitochondria derived from thelymphocytes of a maternally genetically related donor are incubated in4000 Kunitz units of DNase I at 37° C. for 1 hour to eliminate anyresidual DNA of the nucleus. The treated mitochondria are subsequentlywashed through a series of sucrose gradients to remove the DNase Iactivity. Following DNase I treatment, the mitochondrial DNA isextracted from the mitochondria by digesting the mitochondrial membranefor 3 hours at 50° C. with 10 mg/ml proteinase K in 100 mM NaCl, 10 mMEDTA, 50 mM Tris-HCl, pH 7.4 and 1% SDS. The mitochondrial DNA isfurther extracted with phenol and chloroform and precipitated withethanol.

The mitochondrial DNA isolated from the lymphocytes of a maternallygenetically related donor is screened for “normalcy” using completesequence analysis of the mitochondrial genome (see, e.g., Choo-Kang etal. Diabetes 51:2317-2320 (2002), which is incorporated herein byreference). Briefly, 28 to 30 overlapping primers evenly spaced alongthe mitochondrial DNA template are used for polymerase chain reaction(PCR) amplification across the entire 16-17 kilobase mitochondrial DNAgenome. The overlapping PCR fragments are sequenced using an ABI Prism377 DNA Sequencer (from Applied Biosystems, Foster City, Calif.). Theresulting sequence is compared pair-wise using the BLAST2 sequencealignment tool and reference or wild-type sequence information containedin the National Center for Biotechnology Information (NCBI) databasesand/or the MITOMAP database (see e.g. Altschul, et al. J. Mol. Biol.215:403-410 (1990); Ruiz-Pesini et al. Nucleic Acids Res. 35:D823-D828(2007), each of which is incorporated herein by reference).

The complete mitochondrial DNA sequences of a number of mammalianspecies including human, mouse, rat, dog, cat, cow, sheep and horse areavailable in the NCBI databases. In addition, specific informationregarding polymorphisms and mutations of the human mitochondrial genomecan be accessed through the MITOMAP database.

Myoblast cells for autologous transplantation are derived from skeletalmuscle biopsy samples taken from the subject with heart failure (see,e.g. Dib, et al. Circulation 112:1748-1755 (2005), which is incorporatedherein by reference). The skeletal muscle biopsy sample is trimmed ofconnective tissue, minced into a slurry, and digested at 37° C. withtrypsin/EDTA (0.5 mg/ml trypsin, 0.53 mmol/L EDTA) and collagenase (0.5mg/ml) to release the myoblast cells. The myoblast cells are plated andgrown in myoblast basal growth medium (SkBM Basal Medium, Lonza, BaselSwitzerland), containing 15% fetal bovine serum, recombinant humanepidermal growth factor (10 ng/ml), and dexamethasone (3.9 ug/ml). Themyoblast cells are expanded for 11 to 13 doublings prior to harvest.

Mitochondrial DNA is introduced into the myoblast cells usingprotein-mediated transfection (see, e.g., Keeney, et al. Hum. GeneTher., Abstract; 20:897-907 (2009), which is incorporated herein byreference). Briefly, the mitochondrial DNA isolated from themitochondria of the maternally genetically related cells is combinedwith recombinant fusion protein complex containing mitochondrialtranscription factor A (TFAM) engineered to include an N-terminal,11-arginine protein transduction domain followed by a mitochondriallocalization signal (MTD). The recombinant MDT-TFAM protein complexbinds the donor mitochondrial DNA and is used to selectively import, theDNA into the mitochondria of the myoblast cells. MDT-TFAM is combined in2 to 3 fold excess with the donor mitochondrial DNA in a buffer designedto stabilize long DNA templates such as, for example, 85 mM potassiumacetate, 25 mM Tricine (pH 8.7), 8% glycerol, 1% DMS) and 1.1 mMmagnesium acetate and incubated for 30 minutes at 37° C. TheMDT-TFAM-mitochondrial DNA solution is diluted in the myoblast basalgrowth medium described above and applied to the myoblast cells for 4hours at 37° C.

Alternatively, the donor mitochondrial DNA can be introduced into themitochondria of the myoblast cells using a mitochondrial leader sequencepeptide that is recognized by the mitochondrial protein importmachinery. For example, the mitochondrial DNA can be linked to thepre-sequence peptide of the nucleus encoded cytochrome c oxidase (COX)subunit VIII, (U.S. Patent Application 2004/0192627 A1, which isincorporated herein by reference). The mitochondrial DNA is conjugatedto the pre-sequence peptide using pGeneGRIP™-PNA dependent chemistry(from, e.g., Genlantis, San Diego, Calif.). The PNA is designed tohybridize with a portion of the mitochondrial DNA as well as thepre-sequence peptide of COX subunit VIII. In this manner, thepre-sequence peptide is linked to the mitochondrial DNA and canfacilitate import of the DNA into the mitochondria.

The mitochondrial DNA is imported into myoblast cells that have beenpretreated with a mitochondrial toxin as described in Example 1 togenerate ρ⁰ myoblast cells. Alternatively, the mitochondrial DNA isimported into untreated myoblast cells to dilute the endogenousdysfunctional mitochondrial DNA with healthy mitochondrial DNA.

For transplantation, the autologous myoblast cells modified withmitochondrial DNA from the maternally genetically related cells areinjected into the infarcted area of the recipient's heart. One to fourdoses of 1 to 30×10⁷ cells are injected in 3 to 30 injections of 0.1 mleach in a non-overlapping pattern in the area of infarction on theepicardial surface. Transplantation of autologous myoblast cellsmodified with donor mitochondrial DNA can be performed during concurrentcoronary artery bypass grafting (CABG) or left ventricular assist device(LVAD) implantation. See, e.g. Dib, et al. Circulation 112:1748-1755(2005), which is incorporated herein by reference.

Example 3 Treatment of Cytoplasmic Male Sterility in Zea mays UsingMitochondria from Maternally Genetically Related Cells

A method is described for treating cytoplasmic male sterility in Zeamays (maize) using mitochondria isolated from maternally geneticallyrelated cells. Cytoplasmic male sterility is characterized by theinability of an otherwise normal plant to develop functional anthers,pollen, or male gametes, resulting in total or partial male sterility.Cytoplasmic male sterility is associated with a variety of mitochondrialDNA arrangements arising from low frequency, illegitimate recombination,or nonhomologous end joining activity within the mitochondrial genome,resulting in aberrant open reading frames comprised of chimericsequences. (See, e.g., Mackenzie & McIntosh, Plant Cell, 11:571-585,(1999); Kubo & Newton, Mitochondrion; Abstract; 8:5-14(2008), each ofwhich is incorporated herein by reference). In order to restore malefertility in a deficient genotype of Zea mays, mitochondria frommaternally genetically related cells isolated from a genotype of Zeamays exhibiting normal fertility are introduced into developing Zea maysembryos.

Mitochondria for microinjection into Zea mays embryos are isolated fromseedlings of a maternally genetically related genotype. Briefly, onehundred grams of seedlings are placed in a grinding buffer (e.g., 0.35 Msorbitol, 50 mM Tris pH 7.6, 5 mM EDTA, 0.2% bovine serum albumin, 1.0%polyvinylpropyline, 0.025% spermine, 0.025% spermidine, 0.125%betamercaptoethanol) and homogenized in a blender on high for 3×15seconds. Homogenate is filtered through cheesecloth and centrifuged at3,000×g for 10 minutes. The resulting supernatant is centrifuged at16,000×g for 30 minutes. The pellet is gently resuspended in a washbuffer (e.g., 0.35 M sorbitol, 50 mM Tris pH 7.6, 0.1% bovine serumalbumin) and recentrifuged at 16,000×g. The pellet is resuspended inwash buffer with the addition of 25 mM MgCl₂ and treated with 100 ug/mlDNase I for 30-60 minutes on ice to remove contaminating DNA of thenucleus. The suspension is centrifuged at 2,000×g and the resultingsupernatant centrifuged at 16,000×g. The pellet is resuspended in washbuffer and overlayed onto a Percoll gradient and centrifuged at 12,000×gfor 1 hour. The mitochondria are collected at the 23:40 interface,washed with buffer, and centrifuged at 20,000×g for 30 minutes. Thepellet containing purified mitochondria is resuspended in wash bufferand either used immediately to extract DNA or RNA or it is frozen at−20° C. in a 1:1 mixture of wash buffer and 50% glycerol.

The mitochondrial DNA isolated from the maternally genetically relatedcells is isolated using a cesium gradient. Briefly, the mitochondria areresuspended in a lysis buffer (e.g., 1.0% Sarkosyl, 50 mM Tris pH 8.0,25 mM EDTA, 10 mg/ml proteinase K) and digested at 37° C. for 1 hour.Cesium chloride (100% w/v) is dissolved into the digest and ethidiumbromide added to 0.2 mg/ml. The solution is centrifuged at 55,000 rpmfor 20 hours. An ethidium bromide stained band containing themitochondrial DNA is removed and the DNA precipitated with ethanol.

The mitochondrial DNA from the maternally genetically related cells isassessed for “normalcy” using a shotgun sequencing approach (see, e.g.,Allen, et al., Genetics 177:1173-1192 (2007), which is incorporatedherein by reference). The purified mitochondrial DNA is fragmented,ligated into a plasmid cloning vector and transfected into anEscherichia coli host strain (e.g., DH10B-T1) to generate a sequencinglibrary. The fragments of mitochondrial DNA are isolated and sequencedusing an ABI Prism 377 DNA Sequencer (from Applied Biosystems, FosterCity, Calif.). Sequence associated with the plasmid cloning vector issubtracted and overlapping mitochondrial DNA sequences are piecedtogether to generate a complete mitochondrial genome sequence. Theresulting mitochondrial genomic sequence from the maternally geneticallyrelated genotype is compared with that of other fertile strains of Zeamays (e.g., NA and NB) using a sequence alignment tool (e.g., Multalin;Corpet, Nucl. Acids Res., 16:10881-10890, 1988, which is incorporatedherein by reference).

Fertilized embryos for use in microinjection are isolated from Zea maysplants grown to maturity. Methods to isolate plant embryos are known inthe art (see e.g., U.S. Pat. No. 6,300,543, which is incorporated hereinby reference). Maize plants with silks 6-10 cm in length are pollinatedby hand and the plants are placed in a growth chamber for at least 16hours to allow fertilization to occur. Ovaries are isolated by removinghusks and silks from the cobs and cutting the cobs transversely in 3 cmsegments. The segments are sterilized for 10 minutes in 70% ethanol andrinsed in deionized water. Ovaries are then removed and mounted forsectioning.

Specimen blocks for use in the microtome are surface sterilized in 70%ethanol for 10 minutes, and the alcohol is evaporated in a laminar flowhood. A thin layer of adhesive, “Quik Set 404” (available from LocktiteCorp., Newington, Conn.), is used to immobilize the ovaries with theiradiaxial surface up. The blocks with attached ovaries are placed in aVibratome (available from Technical Products International., St. Louis,Mo.), and sectioned at a thickness of 200-400 μm. Microscopic inspectionof the sections is used to identify 250 μm to 300 μm slices containingembryo sacs. Sections containing embryo sacs are collected and placed ona modified Murashige-Skoggs medium with 0.4 mg/L L-asparagine, 0.1 mg/L6-benzylaminopurine (BAP) and 15% sucrose, pH 5.8. Media and cultureconditions for plant embryos are known in the art (see e.g., Hecht etal., Physiol. Plant 127: 803-816 (2001), which is incorporated herein byreference).

Zygotes contained in intact embryo sacs are microinjected with themitochondrial isolated from the maternally genetically related strain.For microinjection, one to six picoliters of solution containing200-2000 mitochondria is microinjected into single oocytes using a StemiSV11 Stereomicroscope (available from Carl Zeiss Microimaging, Inc.,Thornwood, N.Y.) equipped with Narishige micromanipulators and a PL-100pico-injector (micromanipulators and injector are available from TritechResearch, Los Angeles, Calif.). Embryo sacs are cultured at 25° C. inthe dark on modified Murashige-Skoggs medium with 0.4 mg/L L-asparagine,0.1 mg/L 6-benzylaminopurine (BAP) and 15% sucrose, pH 5.8.

In order to grow plants from the microinjected zygotes, the embryo sacsare transferred to media lacking BAP, and cultured in vitro. Embryo sacsare cultured in Murashige-Skoggs medium with 0.4 mg/L L-asparagine and10% sucrose, pH 5.8 for 5 days at 25° C. in the dark, and thentransferred to the same medium containing only 3% sucrose for another 5days. When young shoots are approximately 1.5 cm long, they are exposedto light. After the embryos have grown into plantlets in vitro, they areplanted in sterilized soil or vermiculite and grown to maturity in agreenhouse. The resulting plants are assessed for the presence ofanthers, pollen and/or male gametes as indicators of recovery of malefertility. Various methods for determining viability of pollen and/ormale gametes have been described (see, e.g., Dumas & Mogensen, PlantCell, 5:1337-1348 (1993) which is incorporated herein by reference).

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

What is claimed is:
 1. A method, comprising: extracting DNA from themitochondria of a first vertebrate cell, at least partially based onselecting the Major Histocompatibility type of the first vertebratecell; and transferring the extracted mitochondrial DNA in vitro, as wellas one or more of DNA from the cell nucleus or an entire cell nucleus,from the first vertebrate cell to a second vertebrate cell, wherein thefirst vertebrate cell and the second vertebrate cell are maternallygenetically related to each other.
 2. The method of claim 1, wherein themitochondrial DNA includes one or more mitochondrial chromosomes.
 3. Themethod of claim 1, wherein the mitochondrial DNA is selected bysearching at least one database including information related togenotyped mitochondrial DNA.
 4. The method of claim 1, wherein themitochondrial DNA is selected from a subject source of the firstvertebrate cell that is chronologically younger in age than the subjectsource of the second vertebrate cell.
 5. The method of claim 1, whereinthe mitochondrial DNA is selected at least partially based on the numberof mutations in the mitochondrial DNA as determined by a peptide nucleicacid oligomer probe.
 6. The method of claim 1, wherein the mitochondrialDNA from the first vertebrate cell is transferred to at least oneintracellular target of the second vertebrate cell.
 7. The method ofclaim 6, wherein the at least one intracellular target includes at leastone of a nucleolus, nucleus, ribosome, vesicle, rough endoplasmicreticulum, Golgi apparatus, cytoskeleton, smooth endoplasmic reticulum,mitochondria, vacuole, cytoplasm, lysosome, or centriole.